Much brain disease research focuses on neuronal mechanisms of toxicity. However, glia comprise the most abundant class of cells in the nervous system and little is known about the functional role of these cells in disease states. Mutations in the gene encoding the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), cause Alexander disease, a typically childhood disorder that manifests with seizures and severe white matter pathology. Dysmyelination is accompanied by the formation of GFAP-rich inclusions in astrocytes termed Rosenthal fibers. The presence of GFAP in these aggregates and the observation that overexpression of GFAP in mouse astrocytes produces a severe neurological syndrome and Rosenthal fiber formation has led to the hypothesis that Alexander disease is produced by a dominant gain of function mechanism, perhaps related to abnormal aggregation of GFAP. To create a model of Alexander disease, we have expressed normal and Alexander disease-linked mutant versions of GFAP in Drosophila. We find that overexpression of GFAP in Drosophila glia leads to formation of numerous GFAPcontaining, Rosenthal-fiber like inclusion bodies, increased seizure frequency, and non-cell autonomous neurodegeneration. Preliminary genetic analysis suggests that protein misfolding and oxidative stress play key roles in mediating toxicity of GFAP in our model. Further, we see robust upregulation of autophagy and the JNK pathway in our transgenic animals. Protein misfolding, oxidative stress, JNK pathway activation and autophagy have been implicated in Alexander disease and Alexander disease models in mice and mammalian cell culture systems by other members of the Program Project. Thus, our model appears to identify relevant pathways of GFAP toxicity. We have also developed a screening assay suitable for forward genetic analysis in our model. We now propose a large-scale forward genetic analysis of GFAP cellular toxicity. Our experiments will utilize a newly created whole genome wide transgenic RNAi collection. Novel insights emerging from our work can be further evaluated in biochemical, cellular, and transgenic mouse experiments working with other members of the Program Project. PUBLIC HEALTH RELEVANCE: Alexander disease is a devastating disorder that attacks glia and neurons in the brains of patients, leading to severe disability and early death. Our studies will develop and utilize powerful models of the disease that can be used to understand the precise cellular and biochemical abnormalities that occur, and thus pave the way for effective therapies.